Experimental Study Of Tangshen Formula Improved Renal Fibrosis And Lipid Accumulation In Diabetic Nephropathy

Diabetic nephropathy(DN)has become the main cause of end stage renal disease(ESRD),which challenges the modern global health care.The main pathological changes of DN are the excessive deposition of extracellular matrix(ECM)and the increase of the thickness of glomerular basement membrane(GBM),which leads to the proliferation and expansion of mesangial and eventually to renal fibrosis.Both genetic and environmental factors are linked to the initiation and progression of DN,including genetic pre-disposition,sedentary lifestyle,hypertension,persistent hyperglycemia and dyslipidemia.Although considerable progress has been made in elucidating the molecular mechanisms involved in DN,adequate treatment options for this disease remain limited.Taking advantage of the multi-target treatment of traditional Chinese medicine,it is necessary to focus on the development of traditional Chinese medicine to improve blood lipid disorders,inhibit renal fibrosis and improve renal lipid deposition.Prof.Li proposed Tangshen formula(TSF)based on predecessors experience and clinical experience in order to make the treatment of diabetic kidney diseases(DKD).Multi-center randomized controlled trial study showed which effectively reduced the 24 h urine protein and increased glomerular filtration rate.Animal experiments also found that TSF lowered the levels of serum TC and TG in OLETF rats.However,whether TSF could effectively improve renal fibrosis and lipid deposition in db/db mice with type 2 diabetes and its related mechanisms remain unclear.In our study,we investigated the role of TSF on the modulation of renal fibrosis and lipid deposition in db/db mice.Aims:1.Use of db/db mice to observe the regulation of renal kidney fibrosis and lipid deposition by TSF.2.Investigate potential mechanisms via mTECs cells stimulated by sodium palmitate(PA).Methods:Eight weeks old db/db and db/m mice were randomly divided into three subgroups,(db/m,db/db,db/db+TSF),and all mice were fed with a normal diet.After two weeks adaptation,mice were maintained on TSF treatment for 12 weeks and were then sacrificed under anesthesia after overnight fast.Body weight was recorded weekly.Animal blood and renal tissues samples were collected for further analyses.The kidney washed with cold PBS solution;one part of the renal tissue stored in liquid nitrogen,another deposited-80℃ directly for subsequent frozen sections;the rest of the kidney fixed into the neutral formalin for pathological analysis.PAS and Masson staining investigated the renal fibrosis.Oil red O and Filipin staining investigated renal lipid accumulation.The renal fibrosis expression of TGF-β1/Smad3 and its target genes and renal lipid accumulation expression of PGC-la/PPARa and PGC-la/LXR/ABCA1 were assessed by Western Blotting,real-time PCR,and IHC.The expression of PGC-1α/LXR/ABCAl were assessed by Western Blotting,real-time PCR In sodium palmitate(PA)-stimulated and Abcal-SiRNA-transfected mouse tubular epithelial cells(mTECs).Results:1.TSF reduced dyslipidemia and renal injury in db/db mice.TSF diminished body weight and UACR of db/db mice.Compared with db/m mice,serum levels of LDL-C,HDL-C,TC,and TG were significantly increased in db/db mice mice and were decreased in those treated with TSF,although the level of HDL-C was not significantly affected by the treatment with TSF.Histological analysis using PAS and Masson staining revealed the occurrence of mesangial matrix expansion and extracellular matrix deposition in the kidneys of db/db mice.The treatment with TSF significantly ameliorated these histological renal injuries in db/db mice.2.TSF attenuated renal fibrosis in db/db mice.Western Blotting and real-time PCR analysis showed that expression levels of TGF-β1、Smad3、CollagenIand Fibronectin were significantly downregulated with TSF treatment in the db/m mice.With TSF treatment,the expression levels of miRNA 21 was downregulated,but the expression levels of miRNA 29b was upregulated.3.TSF attenuated renal lipid accumulation in db/db mice.Oil Red O and Filipin cholesterol staining revealed the occurrence of lipid and cholesterol accumulation in the kidneys of db/db mice,and treatment with TSF significantly prevented lipid and cholesterol accumulation.Western Blotting and real-time PCR analysis showed that expression levels of PPARα、PGC-1α、LXR and ABCA1 were significantly downregulated with TSF treatment in the db/db mice.4.TSF promoted renal cholesterol efflux by enhancing the expression of PGC-la,LXR,and ABCA1 in PA-stimulated mTECs.Western Blotting and real-time PCR analysis showed that expression levels of PGC-1α、LXR and ABC A1 were significantly downregulated with TSF treatment in PA-stimulated mTECs.The results of the colorimetric total cholesterol analysis showed that silencing ABCA1 significantly suppressed the inhibitory effect of TSF on PA-induced total cholesterol levels in mTECs without altering protein and mRNA expression levels of PGC-1α and LXR.Conclusions:TSF attenuated renal fibrosis in kidneys of db/db mice,through the downregulation of TGF-β1/Smad3 pathway,and decreased the lipid and cholesterol accumulation in kidneys,through the upregulation of PPARa and ABCA1 in db/db mice.The promotive action of TSF on renal fibrosis and lipid accumulation might contribute to the therapeutic effect in DN.